Glis1 and Glis2 are novel genes recently identified in our laboratory. The Glis1 and -3 genes encode an 84.3 kD and 55.8kD proline-rich protein, respectively. Their five tandem zinc finger motifs exhibit highest homology with those of members of the Gli and Zic subfamilies of Kruppel-like proteins. Glis1 and -2 were mapped to mouse chromosome 4C6 and 16A3-B1, respectively. Northern blot analysis showed that expression of the 3.3 kb Glis1 mRNA is most abundant in placenta and adult kidney and expressed at lower levels in testis. Glis2 was most highly expresed in kidney. Whole mount in situ hybridization on mouse embryos demonstrated that Glis1 is expressed in a temporal and spatial manner during development; expression was most prominent in several defined structures of mesodermal lineage, including craniofacial regions, branchial arches, somites, vibrissal and hair follicles, limb buds, and myotomes suggesting a role at different stages of development. Glis2 was expressed in kidney and neural tube suggesting a role in neurogenesis and kidney development.Confocal microscopic analysis showed that Glis1 and -2 are localized to the nucleus. The zinc finger region plays an important role in the nuclear localization of these proteins. Electrophoretic mobility shift assays demonstrated that Glis1 is able to bind oligonucleotides containing the Gli-binding site consensus sequence GACCACCCAC. Although monohybrid analysis showed that in several cell types Glis1 and -2 are unable to induce transcription of a reporter, deletion mutant analysis revealed the presence of a strong activation and repressor functions. Constitutively active Ca2+-dependent calmodulin kinase IV enhanced Glis1-mediated transcriptional activation about 4-fold and may be mediated by phosphorylation/activation of a co-activator. Our results suggest that Glis1 and -2 may play a critical role in the control of gene expression during specific stages of embryonic development.